Rotina para o exame do semen: resultados encontrados em 30 exames normais / Basic routine for semen analysis: results found in 30 normal examinations

Uma rotina para o exame do semen (espermograma), adaptada a partir dos procedimentos tecnicos recomendados pela Organizaçao Mundial de Saude e descrita neste estudo. Esta rotina abrange o exame fisico (coagulaçao, tempo de liquefaçao, volume ejaculado, cor-aspecto, viscosidade e pH), dosagens bioquimicas (fosfatase acida e frutose), a analise das caracteristicas gerais dos espermatozoides (contagem celular, viabilidade, motilidade e morfologia) e a pesquisa de anticorpos antiespermatozoides. Usando esta rotina, resultados encontrados em 30 semens normais sao apresentados

Actividad de sialiltransferasa en la membrana espermática y su relación con fertilidad y esterilidad humanas / Sialyltransferase activity on spermatic membrane and its relationship with human fertility and sterility

La actividad de la sialiltransferasa (ST) fue determinada en los espermatozoides (spz) de sujetos normospérmicos (>80 X 10 6 spz/ml y mn[as del 75% de movilidad) y en los spz de pacientes con problemas de fecundidad (oligospérmicos < 20 y 10 6 spz/ml y menos del 20% de movilidad y astenospérmicos > 40 X 10 6 spz/ml y menos del 10% de movilidad). La actividad de la ST se cuantifica mediante la transferencia de radiactividad de CMP -3 H- siálico hacia el aceptor exógeno (fetuina desializada). El complejo enzima sustrato formado, al ser colocado en presencia del ácido fosfotúrgstico da un producto de fosfotungstato insoluble, el cual es retenido en un filtro de fibra de vidrio. La actividad enzimática disminuye en los spz de oligospérmicos en un 62 + - 5% con respecto a los spz de normospérmicos. La disminución de la actividad de la ST en los spz de pacientes infértiles permite suponer que esta enzima participa probablemente como causa directa de su patología y que su disminución obedece a un daño en la integridad estructural de la membrana espermática.(au)

Solubilizacion de la membrana del espermatozoide humano con n-dodecil sarcosinato de sodio: I.accion del detergente / Solubilization of the membrane human spermatozoa: I. action of the detergent

Espermatozoides humanos fueron lavados con PBS y tratados com n-dodecil sarcosinato de sodio (Sarkosyl). El insoluble remanente estaba compuesto por cabezas, cuyas colas fueron cortadas a la altura del cuello o del segmento intermedio y cuyas membranas no sufrieron mayores danos, estando el acrosoma intacto y por pequenos corpusculos que podrian ser acrosomas aislados. Los resultados indican que la accion del detergente es de dos tipos claramente diferenciados: a)diseccion del espermatozoide con separacion de la cola y b)disolucion del mismo dejando el acrosoma intacto. El residuo conserva actividad inmunologica e inmunobiologica

Espermograma: determinaçao da concentraçao de espermatozoides utilizando as camaras de Neubauer, Makler e Horwell / Semen analysis: comparison of Neubauer, Makler and Horwell counting chambers

Os tres metodos manuais de determinaçao da concentraçao de espermatozoides mais utilizados no espermograma foram comparados. As concentraçoes de espermatozoides de 96 amostras de semen foram obtidas pelos metodos de contagem em camara de Neubauer, de Makler e de Horwell e de acordo com a motilidade e concentraçao dos espermatozoides, as amostras foram divididas em tres grupos: Normozoospermicas; Astenozoospermicas e Oligoastenozoospermicas. Os resultados obtidos utilizando-se as camaras de Neubauer e Makler nao apresentaram diferenças significativas, enquanto que, com a camara de Horwell os resultados foram significativamente maiores do que os obtidos com as duas outras camaras

Coloraçao supravital para espermatozoides: comparaçao entre as tecnicas de eosina e eosina-nigrosina / Comparison of supravital staining techniques for spermatozoa: eosin and eosin-nigrosin

Neste estudo, 66 amostras de semen foram utilizados para comparar duas tecnicas laboratoriais de coloraçao supravital para espermatozoides. Foram utilizadas eosina a 0,5 por cento nas preparaçoes a fresco e uma soluçao de eosina a 1 por cento e nigrosina a 10 por cento nos esfregaços. Independente da concentraçao da amostra, os resultados obtidos com ambas as tecnicas nao apresentaram diferenças significantes

Immunohistochemical localization of nuclear 3,5,3'-triiodothyronine receptor proteins in rat tissues studied with antiserum against C-ERB A/T3 receptor.

We have previously raised an anti-c-erb A peptide antibody (designated 4B II) which immunoprecipitated in vitro transcription/translation products of c-erb A alpha 1 and beta. 4B II could recognize nuclear T3 receptor (NT3R) without distinction between difference in species and tissues. Using 4B II, we studied immunohistochemical localization of NT3R proteins in various tissues of the rat. Cryostat sections (4-6 microns) of selected rat tissues were incubated with 4B II at 4C overnight, followed by fluorescein-isothyocianate-conjugated anti-rabbit immunoglobulin G for 60 min at 25 C. The cellular localization of fluorescence in all tissues examined was exclusively nuclear. Under the same conditions, control sections stained with antiserum which had previously absorbed with c-erb A peptide or inactive serum showed no specific staining. In the brain the large nuclei, supposed to be neuronal, were strongly stained in the cerebral cortex and the granular layer of the cerebellum. In the kidney, cells in the glomerulus, the distal, but not the proximal, tubules, and the collecting ducts exhibited nuclear staining. Nuclear fluorescence was observed homogeneously in the heart and liver, but the intensity was much weaker in the latter. Less intense fluorescence was seen in the testis and spleen, although specific immunostaining was clearly observed in the nuclei of spermatocytes, Leydig cells, and the heads of the sperms in the testis, and many lymphocytes in the spleen. Nuclei of follicular cells of the thyroid exhibited very strong fluorescence, suggesting existence of plenty of NT3R proteins. The anterior pituitary showed strong immunostaining in most nuclei, and clear nuclear fluorescence was also detected in the intermediate lobe of the pituitary. The present study showed that NT3R distributes selectively in certain types of cells in many tissues and that the content of NT3R proteins seems to correlate with the concentration of c-erb A mRNA alpha 1 and beta among many organs.

Lipid domains in the ram sperm plasma membrane demonstrated by differential scanning calorimetry.

Mammalian sperm plasma membranes, in contrast to those of mammalian somatic cells, exhibit a significant fraction of lipid that does not diffuse laterally in the plane of the membrane. This nondiffusing fraction results from lipid-lipid interactions. Similar nondiffusing fractions are found in mixed-lipid model systems that contain coexistent gel and fluid domains. These results suggest that the sperm plasma membrane may also exhibit lateral phase segregations of lipids and may contain significant amounts of gel-phase lipid. In this paper we use differential scanning calorimetry to show that, in contrast to the plasma membranes of mammalian somatic cells, the plasma membrane from the anterior region of the head of ram sperm exhibits at least two major endothermic transitions, one centered at approximately 26 degrees C and one centered at approximately 60 degrees C. The heats of these transitions are consistent with gel-to-fluid transitions in model membranes. These transitions are observed both in plasma membrane vesicles and in rehydrated lipid extracts made from these vesicles. These results demonstrate that at physiological temperatures the lipids of the ram sperm plasma membrane are segregated into coexistent fluid and gel domains. Since sperm encounter a wide range of temperatures during their development, these phase transitions may be important in establishing dynamic domains of lipid requisite for epididymal storage and fertilization.

Optimum testicular aspiration technique for deoxyribonucleic acid flow cytometric analysis of spermatogenesis in primates (Papio anubis).

Deoxyribonucleic acid flow cytometry on testicular tissue is an established method to evaluate spermatogenesis. Needle aspiration is less invasive and permits sequential sampling as compared with open testis biopsy. Using seven primates, we examined the needle aspiration technique. There was no significant difference among locations aspirated; the Westcott needle (Bard Urological, Covington, GA) provided the most rapid analysis, and both the Biopty gun (Bard Urological) and Westcott needle were easy to use. Baseline means +/- SD for ploidy distributions were 1N: 65.73% +/- 7.1; 2N: 18.95% +/- 5.5; 4N: 11.4% +/- 2, Experimental data generated from primate models using testis aspiration and flow cytometry may elucidate human infertile conditions.

Measurement of DNA breakage in specific germ-cell stages of male mice exposed to acrylamide, using an alkaline-elution procedure.

DNA breakage in spermiogenic stages of the mouse was studied after exposure to acrylamide (AA), using an alkaline-elution technique. At daily intervals over a 3-week period following i.p. injection of 100 mg AA/kg, mature spermatozoa were recovered from treated ([3H]dThd-labeled) and control ([14C]dThd-labeled) animals, and were lysed together on polycarbonate filters; the DNA was eluted with a high-pH (12.0) buffer. Elution of germ-cell DNA from AA-exposed animals increased (more DNA-strand breaks) in stages sensitive to the dominant-lethal effects of AA (late spermatids to early spermatozoa) (Shelby et al., 1986). The stage-related pattern of AA-induced DNA breakage also paralleled the pattern of sperm alkylation and protamine alkylation found to be produced by AA (Sega et al., 1989). While dominant-lethal damage from AA exposure is greatest in the spermatids and early spermatozoa, no such damage was observed in pachytene spermatocytes and early spermatids (Shelby et al., 1986). Therefore, AA-induced DNA breakage was also studied directly in pachytene spermatocytes and in early spermatids at short intervals (up to 4 days) after exposure. DNA breakage was clearly detected in these cell stages, with maximum breakage occurring at 1 day after treatment. At later times, the breakage gradually decreased, presumably as a result of DNA repair. By the time these cell stages gave rise to functional spermatozoa, DNA breaks that could have produced dominant-lethal events had apparently been reduced to a level where no genetic effect could be observed.

Estudio citogenético, bioquímico y de la función reproductiva en personas expuestas a plaguicidas / Cytogenetic, biochemist and reproductive function studie in exposed biocides

El intercambio de cromátides hermanas (ICH) (sensible indicador de mutágenos ambientales), el hemograma, la transaminasa glutámico oxalacética (TGO), la transaminasa glutámico pirúvica (TGP), la colinesterasa, los organoclorados y organofosforados en sangre periférica y el esperma, fueron estudiados en 44 individuos expuestos a plaguicidas y 36 controles no expuestos. El grupo de individuos expuestos estaba constituido por trabajadores agropecuarios en contacto directo o indirecto con los plaguicidas (organoclorados y organofosforados). Los estudios a este grupo se realizaron en 3 épocas distintas. Para cada una de las variables en consideración, se realizaron comparaciones expuestos y controles mediante el test "T" de Student. Se encontró significativo incremento en los eosinófilos y organoclorados, disminución en los glóbulos blancos del grupo expuesto con respecto al control; en el esperma se observaron alteraciones en la movilidad. En los ICH, niveles de TGO, de TGP, organoclorados y colinesterasa, no se detectaron diferencias significativas con respecto a los controles. Este trabajo es un enfoque global al problema y de ninguna manera pretende considerarse definitivo, se continuará profundizando en los diversos aspectos que lo constituyen.

Molecular characterization of nuclear basic protein HPI1, a putative precursor of human sperm protamines HP2 and HP3.

The largest intermediate basic protein HPI1 (101 residues) from human sperm chromatin was isolated and characterized. The amino acid composition and sequence analysis of the protein and of tryptic peptides together with peptide mapping of endoproteinases Lys-C and Glu-C hydrolysates showed that the C-terminal region (residues 45-101) of HPI1 is identical to protamine HP2. These structural data strongly suggest that protein HPI1 is a precursor of human sperm protamines HP2 and HP3 (57 and 54 residues, respectively) as well as of two other intermediate basic proteins HPS1 and HPS2 (69 and 66 residues, respectively) sequenced previously.

Immunocytochemical localization of nuclear protamine in boar spermatozoa during epididymal transit.

Protamine was specifically demonstrated in boar spermatozoa collected from the rete testis, caput, corpus and cauda epididymidis and the ejaculate by immunoelectron microscopy, using anti-boar or anti-ram protamine antisera and an indirect post-embedding immunogold technique. Spermatozoa from all collection sites stained after incubation although with different degrees of labelling. Controls were negative. Labelling increased from the rete testis towards the epididymal corpus, where it was most intense, decreasing sharply thereafter. The weakest binding of the assayed antibodies was obtained in the ejaculated spermatozoa but it could be reversed by in-vitro induction of chromatin decondensation with sodium dodecyl sulphate and the metal-chelating EDTA. The finding of a significant decrease in the immunolabelling detected from the corpus epididymidis onwards indicates a critical point for the interaction between DNA and the protamines in boar spermatozoa during the epididymal maturation.

Interactions of labeled epididymal secretory proteins with spermatozoa after injection of 35S-methionine in the mouse.

The sequential interactions of epididymal secretory proteins with spermatozoa during epididymal transit were examined. Mice received injections of 35S-methionine, and the radiolabeled luminal fluid and sperm-associated proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis at various times after injection. The majority of the luminal fluid and sperm-associated proteins were found in the caput epididymidis at 8 h; by 7 days, many of these proteins had been transported to the cauda epididymidis. Two classes of epididymal protein-sperm interactions were distinguished on the basis of regional synthesis and secretion. The major class consisted of proteins that were synthesized, secreted, and bound to spermatozoa in the caput epididymidis. In this class, however, the binding of proteins to the spermatozoa was variable. For example, a protein of 25 kDa remained associated with spermatozoa in substantial amounts during epididymal transit, while proteins of 40 and 35 kDa decreased in amount. Other proteins such as a protein of 18 kDa did not remain associated with spermatozoa. Another class of proteins (54, 44, 29 kDa) were synthesized and secreted from all epididymal regions but bound only to caput spermatozoa. Most of the epididymal proteins appeared to be tightly bound to the spermatozoa since spermatozoa already saturated with the unlabeled protein in the distal epididymis remained so even though the spermatozoa were surrounded by labeled proteins in the luminal fluid. These studies demonstrate that a variety of specific interactions occur between epididymal secretory proteins and spermatozoa as they migrate and mature in the epididymis.

Identification and characterization of a testis-specific isoform of a chaperonin in a moth, Heliothis virescens.

Two relatively abundant proteins having subunit molecular weights of 60,000 and 63,000 (p60 and p63, respectively) have been purified as a 16 to 18S complex from sperm mitochondria of a moth. Heliothis virescens. Although the function of these proteins had heretofore not been established, interest in the p63 polypeptide stemmed from its sperm-specific expression and its striking occurrence as a net charge variant among several insect species surveyed, using two-dimensional gel electrophoresis. Genomic and cDNA clones corresponding to the p63 protein have now been isolated and their sequencing has revealed extensive amino acid sequence identity with both the Escherichia coli GroEL protein and its eukaryotic homologues, the chaperonins. Immunoblot studies with a Tetrahymena chaperonin antiserum demonstrated that the p60 protein, which is expressed in all cell types, is structurally related to p63 and is itself a chaperonin subunit. While the chaperonin complex from Heliothis sperm shares certain properties with GroEL, including the ability to hydrolyze ATP and organization of its subunits into a seven-member ring, electron microscopic analysis revealed that its higher-order structure differed from GroEL (and other lower eukaryotic chaperonins) in that the native particle comprises one such ring rather than a doublet. It is not yet known whether the two chaperonin isoforms coexpressed in moth sperm assemble separately or give rise to hybrid particles. In either case, the existence of multiple chaperonin subunits in sperm leaves open the possibility that some aspect of mitochondrial biogenesis that is dependent upon the activity of these proteins is qualitatively or quantitatively different in this cell type.

High-resolution proton nuclear magnetic resonance characterization of seminolipid from bovine spermatozoa.

The high-resolution one- and two-dimensional proton nuclear magnetic resonance (1H-NMR) characterization of seminolipid from bovine spermatozoa is presented. The 1H-NMR data was confirmed by gas-liquid chromatography-mass spectrometric analysis of the partially methylated alditol acetates of the sugar unit, mild alkaline methanolysis of the glyceryl ester, mobility on normal phase and diphasic thin-layer chromatography (HPTLC), and fast atom bombardment mass spectrometry (FAB-MS). The structure of the molecule corresponds to 1-O-hexadecyl-2-O-hexadecanoyl-3-O-beta-D-(3'-sulfo)-galactopyranosyl- sn-glycerol.

Evidence for a calsequestrin-like calcium-binding protein in human spermatozoa.

Human spermatozoa were investigated for the presence of protein(s) recognized by antibodies against calsequestrin, the high capacity, moderate affinity Ca2(+)-binding protein, originally described in striated muscle fibers. Western immunoblots of detergent-soluble sperm extracts probed with polyclonal antibodies raised against human skeletal muscle calsequestrin identified a strongly cross-reactive protein. This protein resembles muscle calsequestrin in many respects. In fact, its migration in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is pH dependent, its apparent molecular mass being 64 kDa in alkaline SDS-PAGE and 44 kDa in neutral SDS-PAGE; its isoelectric point is acidic (4.6); it is metachromatically stained blue by the carboxycyanine dye, Stains-All; it is a Ca2(+)-binding protein (45Ca blot overlay). Indirect immunofluorescence experiments showed that the immunoreactive protein has an intracellular localization confined to the tail mid-piece. From these findings we conclude that human sperm cells express a protein structurally and antigenically related to skeletal muscle calsequestrin; a basis for a novel interpretation of Ca2(+)-mediated events in spermatozoa is thus provided.

Regional differences in composition of the perforatorium and outer periacrosomal layer of the rat spermatozoon as revealed by immunocytochemistry.

The rat perforatorium is the part of the perinuclear theca that underlies the acrosomic system. It appears to be composed of several polypeptides. The main objective of this study was to determine the distribution of seven of these perforatorial polypeptides in the head of the rat spermatozoon. For this purpose, polyclonal antibodies were affinity purified from these polypeptides and tested 1) for their distribution on electron-microscope sections of late spermatids and spermatozoa by immunogold labeling and 2) for their specificity on Western blots of denatured perforatorial polypeptides by immunoblotting. Immunoblotting showed that all seven of the prominent perforatorial polypeptides had epitopes in common. Immunogold labeling of spermatozoa showed that antibodies against the 13, 13.4, and 16 kDa polypeptides were restricted in their localization to the thicker apical portion of the perforatorium and to the inner zone of the ventral spur. However, antibodies against the 34, 43, 57, and 63 kDa polypeptides reacted with the entire perforatorium but, in addition, reacted with the inner part of the ventral spur and with a portion of the "outer periacrosomal layer" lying between the plasma membrane and the outer acrosomal membrane. These results suggest 1) that there are regional differences in protein composition of the perforatorium, of the outer periacrosomal layer, and of the postacrosomal dense lamina; and 2) that perforatorial polypeptides may not necessarily be restricted to the subacrosomal region, but may also compose portions of the outer periacrosomal layer and postacrosomal dense lamina. Based on both immunoblotting and immunocytochemical results, using an antiactin monoclonal antibody that recognizes all known isoforms of actin, actin was not detected in the perforatorium of step 19 spermatids or spermatozoa. Actin, however, together with the seven perforatorial polypeptides tested, was present in the subacrosomal space of elongating spermatids before the process of condensation of the perforatorium takes place.